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Crystals have different sizes and shapes. Crystals of calcium oxalate (CaOx), monosodium urate (MSU), cystine, calcium phosphate, cholesterol, asbestos, and silica were imaged using scanning electron microscope (scale bar = 10 µm).
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The human body is exposed to a wide range of particles of industrial, environmental or internal origin such as asbestos, alum, silica or crystals of urate, calcium phosphate, calcium oxalate, cystine or cholesterol. Phagocytic clearance of such particles involves neutrophils and macrophages. Here we report that neutrophils encountering such particl...
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... we also examined crocidolite asbestos fibers. Figure 1 illustrates the different sizes and shapes of these particles as cap- tured by scanning electron microscopy. We isolated human neutrophils from healthy blood donors at a purity of >95% (Supplementary Figure 1). ...
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... 1 illustrates the different sizes and shapes of these particles as cap- tured by scanning electron microscopy. We isolated human neutrophils from healthy blood donors at a purity of >95% (Supplementary Figure 1). Exposing such neutrophils to each of these particles for 2 hours induced aggre- gated-NET-like extracellular DNA relase formed by the particles, intact neutrophils, dead neutrophil debris, and their respective extracellular chromatin ( Fig. 2A, Supplementary Figure 2A,B). ...
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... Crystal-induced cell death The interaction between crystals and cells can induce a broad range of immune responses. Crystals are taken up by cells into phagolysosomes where this material is broken down and digested, whereas an overload of indigested crystals will induce cell stress, actin depolymerization, reactive oxygen species (ROS) production, and NLRP3 inflammasome activation [14]. Many crystals are cytotoxic to different cell types by inducing lysosomal rupture, tyrosine phosphorylation, activation of cathepsins, and mitochondrial dysfunction, as well as ET formation in neutrophils and other immune cells (Boxes 2 and 3 and Figure 2) [15]. ...
... Two types of CaOx crystals have been identified in the urine: CaOx monohydrate (dumbbell shaped) and CaOx dehydrate (bipyramid shaped); the former are predominantly found in patients with kidney stones [33]. In CaOx crystal-induced acute kidney injury, neutrophils undergo RIPK1-RIPK3-MLKL-dependent neutrophil necroptosis and/or form NETs [14] upon phagocytosis, and subsequently contribute to necroinflammation and tissue damage. Brushite crystals are frequently found in patients with kidney and urinary stones. ...
... Asbestos can be sensed by the NLRP3 inflammasome leading to IL-1β secretion [70]. Ex vivo experiments showed that asbestos can trigger RIPK1-RIPK3-MLKL-dependent neutrophil necroptosis and NET formation in neutrophils [14]. Silica crystals, however, are considered harmless and non-toxic, although inhalation of silica can cause pulmonary inflammation (silicosis) [20]. ...
... At least in few cases [18], the initiation center provoking those abundant NETs formation were hydrophobic nanoparticles (or microparticles). Both endogenic (cholesterol, monosodium urate, oxalates) or exogenic (soot, silica, asbestos [22], aluminum oxide [23]) nanoparticles tend to interact with neutrophils and function of dealing with particulate pollutants looks to be one of many important neutrophil features. Even natural minerals (clinoptilolite from the Transcarpathian region) when injected into the organism were shown to induce NET formation [24]. ...
... Hydrophobic nanoparticles (or microparticles) have been identified as initiation centers for abundant NET formation [18]. Both endogenic (cholesterol, monosodium urate, oxalates) and exogenic (soot, silica, asbestos [22], aluminum oxide [23]) nanoparticles tend to interact with neutrophils, and dealing with particulate pollutants seems to be one of the essential functions of neutrophils. Even natural minerals, like clinoptilolite from the Transcarpathian region, when injected into the organism, were shown to induce NET formation [24], supporting the concept that passivating localized particles with NETs (and forming aggNETs) is a universal approach that neutrophils employ for every inert nano-/microparticle nondestructible by other means. ...
... NET formation induced by nanoparticles has been verified through phase-contrast fluorescent microscopy, illustrating extracellular nuclear DNA co-localizing with citrullinated histone H3 and cytoplasmic proteases like neutrophil elastase (NE) during neutrophil-nanoparticle interactions [22]. Additionally, some studies have shown that nanoparticle-induced NETs are PAD4and NE-dependent, indicating that histone citrullination by PAD4 and histone degradation by NE occur directly during NET formation [11]. ...
Neutrophil extracellular traps (NETs) have been identified as triggers for a self‐limited inflammatory reaction upon contact with nanoparticles within our bodies. This typically results in entrapping potentially harmful nano‐ or micro‐objects following an immune burst. The demand for potent adjuvants has led to research on particulate‐based adjuvants, particularly those that act via NET formation. Various particles, including hydrophobic nanoparticles, needle‐like microparticles, and other natural and artificial crystals, have been shown to induce NET formation, eliciting a robust humoral and cellular immune response toward co‐injected antigens. The NET formation was found to be the basis of the efficient use of alum as a vaccine adjuvant. Thus, nanoparticles with specific surface properties serve as NET‐stimulating adjuvants. In this mini‐review, we aim to summarize the current knowledge about the surface properties of particulate objects and the molecular pathways involved in inducing NET formation by neutrophils. Additionally, we discuss the potential use of nanoparticles for activating neutrophils in the tissues and the exploitation of such activation for enhancing vaccine adjuvants.
... Large amounts of neutrophils infiltrate synovial cavity and readily form NETs which are the source of extracellular self-antigens generating strong innate and adaptive response in the joints leading to tissue injury [103,[138][139][140]. Citrullinated NETs serve as an autoantigen [138], and high levels of active PAD4 in NETs play a pathogenic role in rheumatoid arthritis [141]. Gout is an inflammatory disease characterized by deposition of monosodium urate crystals in the joints, stimulating immune response by attracting leukocytes and inducing NETosis which ultimately increase the inflammation [103,[142][143][144]. HMGB1 in the NETs obtained from the synovial fluid of gout patients exhibits pro-inflammatory potential [144]. ...
Pancreatitis is an inflammatory disease, which is triggered by adverse events in acinar cells of the pancreas. After the initial injury, infiltration of neutrophils in pancreas is observed. In the initial stages of pancreatitis, the inflammation is sterile. It has been shown that the presence of neutrophils at the injury site can modulate the disease. Their depletion in experimental animal models of the acute pancreatitis has been shown to be protective. But information on mechanism of contribution to inflammation by neutrophils at the injury site is not clear. Once at injury site, activated neutrophils release azurophilic granules containing proteolytic enzymes and generate hypochlorous acid which is a strong microbicidal agent. Additionally, emerging evidence shows that neutrophil extracellular traps (NETs) are formed which consist of decondensed DNA decorated with histones, proteases and granular and cytosolic proteins. NETs are considered mechanical traps for microbes, but there is preliminary evidence to indicate that NETs, which constitute a special mechanism of the neutrophil defence system, play an adverse role in pancreatitis by contributing to the pancreatic inflammation and distant organ injury. This review presents the overall current information about neutrophils and their role including NETs in acute pancreatitis (AP). It also highlights current gaps in knowledge which should be explored to fully elucidate the role of neutrophils in AP and for therapeutic gains.
... NLRP3 inflammasome driven inflammation and several regulated necrosis pathways 140 are triggered in the region around the crystal 141 . Recruited neutrophils will release neutrophil extracellular traps 142 via triggering necroptosis 143,144 . Following this step, granuloma forma tion around the crystal occurs 134,137 along with platelet Calculi in the context of crystal biology, calculi are a dense aggregate of crystals large enough to be seen on radiographs. ...
... 152 Furthermore, patients with CKD present with chronic low-grade inflammation, 8 and IL8 9-11 and HMGB1 11,153 as known drivers of NETs formation have been reported to be increased in blood of patients with CKD. Also, patients with advanced CKD frequently display hyperphosphatemia and have an increased risk of medial calcification, characterized by a deposition of calcium and phosphate in the medial layer of the vasculature 47,154 Calcium phosphate-based particles-along with other crystalline nanoparticles and microparticles-can induce NETs formation by neutrophils 155,156 by increasing ROS production 156 and RIPK1/MLKL (receptor-interacting protein kinase 1/mixed-ineage kinase like)-dependent necroptosis. 155 The contribution of altered platelet reactivity to NET formation in CKD has not yet been investigated, and highly variable results have been obtained in terms of platelet activity in patients with CKD. ...
... Also, patients with advanced CKD frequently display hyperphosphatemia and have an increased risk of medial calcification, characterized by a deposition of calcium and phosphate in the medial layer of the vasculature 47,154 Calcium phosphate-based particles-along with other crystalline nanoparticles and microparticles-can induce NETs formation by neutrophils 155,156 by increasing ROS production 156 and RIPK1/MLKL (receptor-interacting protein kinase 1/mixed-ineage kinase like)-dependent necroptosis. 155 The contribution of altered platelet reactivity to NET formation in CKD has not yet been investigated, and highly variable results have been obtained in terms of platelet activity in patients with CKD. This varies from reduced platelet aggregation and activity over no changes to increased platelet responses in CKD-as discussed in detail elsewhere 121 -with heterogeneity in CKD pathophysiology, severity, or potential comorbidities as potential explanations. ...
Arterial and venous thrombosis constitute a major source of morbidity and mortality worldwide. Association between thrombotic complications and cardiovascular and other chronic inflammatory diseases are well described. Inflammation and subsequent initiation of thrombotic events, termed immunothrombosis, also receive growing attention but are still incompletely understood. Nevertheless, the clinical relevance of aberrant immunothrombosis, referred to as thromboinflammation, is evident by an increased risk of thrombosis and cardiovascular events in patients with inflammatory or infectious diseases. Proinflammatory mediators released from platelets, complement activation, and the formation of NETs (neutrophil extracellular traps) initiate and foster immunothrombosis. In this review, we highlight and discuss prominent and emerging interrelationships and functions between NETs and other mediators in immunothrombosis in cardiovascular disease. Also, with patients with chronic kidney disease suffering from increased cardiovascular and thrombotic risk, we summarize current knowledge on neutrophil phenotype, function, and NET formation in chronic kidney disease. In addition, we elaborate on therapeutic targeting of NETs-induced immunothrombosis. A better understanding of the functional relevance of antithrombotic mediators which do not increase bleeding risk may provide opportunities for successful therapeutic interventions to reduce thrombotic risk beyond current treatment options.
... In contact with the crystals NETs are generated and promote growth of gall stones. Various kinds of crystals, among other sterile stimuli, are potent inducers of neutrophil activation and NET formation [204]. ...
Extracellular chromatin, for example in the form of neutrophil extracellular traps (NETs), is an important element that propels the pathological progression of a plethora of diseases. DNA drives the interferon system, serves as autoantigen, and forms the extracellular scaffold for proteins of the innate immune system. An insufficient clearance of extruded chromatin after the release of DNA from the nucleus into the extracellular milieu can perform a secret task of moonlighting in immune-inflammatory and occlusive disorders. Here, we discuss (I) the cellular events involved in the extracellular release of chromatin and NET formation, (II) the devastating consequence of a dysregulated NET formation, and (III) the imbalance between NET formation and clearance. We include the role of NET formation in the occlusion of vessels and ducts, in lung disease, in autoimmune diseases, in chronic oral disorders, in cancer, in the formation of adhesions, and in traumatic spinal cord injury. To develop effective therapies, it is of utmost importance to target pathways that cause decondensation of chromatin during exaggerated NET formation and aggregation. Alternatively, therapies that support the clearance of extracellular chromatin are conceivable.
... In an in vivo mouse model of oxalate nephropathy, knockout of Ripk3 or Mlkl, knockout of Tnfr1 and Tnfr2, and application of a TNF inhibitor or the RIPK1 inhibitor Nec-1 all improved kidney function 78 . Two additional studies supported the idea that diverse crystalline particles trigger RIPK1/3 and MLKL-dependent necroptosis in neutrophils, resulting in the release of neutrophil extracellular trap (NET)-like extracellular DNA 78,79 . Further studies highlighted the therapeutic relevance of necroptosis inhibition in mouse models of oxalate nephropathy [80][81][82] . ...
... These cells release chromatin fibres called NETs, which are targeted by ANCAs, and undergo a form of lytic cell death termed NETosis 125 . Several studies have reported that neutrophil NET generation is dependent on the necroptosis proteins RIPK1, RIPK3 and MLKL in various contexts 78,79,126 , although other reports have highlighted GSDMD-dependent pyroptosis as a driver of NETosis [127][128][129] . Of particular interest is a 2017 report, which found that ANCA stimulation induced RIPK1-dependent necroptosis of neutrophils, which induced the release of NETs. ...
Cell death, particularly that of tubule epithelial cells, contributes critically to the pathophysiology of kidney disease. A body of evidence accumulated over the past 15 years has ascribed a central pathophysiological role to a particular form of regulated necrosis, termed necroptosis, to acute tubular necrosis, nephron loss and maladaptive renal fibrogenesis. Unlike apoptosis, which is a non-immunogenic process, necroptosis results in the release of cellular contents and cytokines, which triggers an inflammatory response in neighbouring tissue. This necroinflammatory environment can lead to severe organ dysfunction and cause lasting tissue injury in the kidney. Despite evidence of a link between necroptosis and various kidney diseases, there are no available therapeutic options to target this process. Greater understanding of the molecular mechanisms, triggers and regulators of necroptosis in acute and chronic kidney diseases may identify shortcomings in current approaches to therapeutically target necroptosis regulators and lead to the development of innovative therapeutic approaches.
... The cationic CysR5 peptide 1 is unable to initiate NET release because it lacks the hydrophobic character that is needed to disrupt the lysosomal membrane. Furthermore, the neutral silica particles did not promote NET formation and we were able to confirm that particle size and shape do not influence the induction of NET as reported by Desai et al. [86]. ...
Nature offers a wide range of evolutionary optimized materials that combine unique properties with intrinsic biocompatibility and that can be exploited as biomimetic materials. The R5 and RRIL peptides employed here are derived from silaffin proteins that play a crucial role in the biomineralization of marine diatom silica shells and are also able to form silica materials in vitro. Here, we demonstrate the application of biomimetic silica particles as a vaccine delivery and adjuvant platform by linking the precipitating peptides R5 and the RRIL motif to a variety of peptide antigens. The resulting antigen-loaded silica particles combine the advantages of biomaterial-based vaccines with the proven intracellular uptake of silica particles. These particles induce NETosis in human neutrophils as well as IL-6 and TNF-α secretion in murine bone marrow-derived dendritic cells.
... NETosis is considered as a regulated process [56]. NADPH oxidase function is crucial for the formation of NETs [57], and inhibition of peptydilarginine deiminase type 4 or abrogation of reactive oxygen species inhibits NET-dependent gallstone formation in vivo [29]. ...
Nano- and microparticles enter the body through the respiratory airways and the digestive system, or form as biominerals in the gall bladder, salivary glands, urinary bladder, kidney, or diabetic pancreas. Calcium, magnesium, and phosphate ions can precipitate from biological fluids in the presence of mucin as hybrid nanoparticles. Calcium carbonate nanocrystallites also trap mucin and are assembled into hybrid microparticles. Both mucin and calcium carbonate polymorphs (calcite, aragonite, and vaterite) are known to be components of such biominerals as gallstones which provoke inflammatory reactions. Our study was aimed at evaluation of neutrophil activation by hybrid vaterite–mucin microparticles (CCM). Vaterite microparticles (CC) and CCM were prepared under standard conditions. The diameter of CC and CCM was 3.3 ± 0.8 µm and 5.8 ± 0.7 µm, with ƺ-potentials of −1 ± 1 mV and −7 ± 1 mV, respectively. CC microparticles injured less than 2% of erythrocytes in 2 h at 1.5 mg mL−1, and no hemolysis was detected with CCM; this let us exclude direct damage of cellular membranes by microparticles. Activation of neutrophils was analyzed by luminol- and lucigenin-dependent chemiluminescence (Lum-CL and Luc-CL), by cytokine gene expression (IL-6, IL-8, IL-10) and release (IL-1β, IL-6, IL-8, IL-10, TNF-α), and by light microscopy of stained smears. There was a 10-fold and higher increase in the amplitude of Lum-CL and Luc-CL after stimulation of neutrophils with CCM relative to CC. Adsorption of mucin onto prefabricated CC microparticles also contributed to activation of neutrophil CL, unlike mucin adsorption onto yeast cell walls (zymosan); adsorbed mucin partially suppressed zymosan-stimulated production of oxidants by neutrophils. Preliminary treatment of CCM with 0.1–10 mM NaOCl decreased subsequent activation of Lum-CL and Luc-CL of neutrophils depending on the used NaOCl concentration, presumably because of the surface mucin oxidation. Based on the results of ELISA, incubation of neutrophils with CCM downregulated IL-6 production but upregulated that of IL-8. IL-6 and IL-8 gene expression in neutrophils was not affected by CC or CCM according to RT2-PCR data, which means that post-translational regulation was involved. Light microscopy revealed adhesion of CC and CCM microparticles onto the neutrophils; CCM increased neutrophil aggregation with a tendency to form neutrophil extracellular traps (NETs). We came to the conclusion that the main features of neutrophil reaction to mucin–vaterite hybrid microparticles are increased oxidant production, cell aggregation, and NET-like structure formation, but without significant cytokine release (except for IL-8). This effect of mucin is not anion-specific since particles of powdered kidney stone (mainly calcium oxalate) in the present study or calcium phosphate nanowires in our previous report also activated Lum-CL and Luc-CL response of neutrophils after mucin sorption.
... There is growing interest in the synthesis and regulation of NETs. RIPK1-RIPK3-MLKL-dependent neutrophil necroptosis has previously been associated with NET formation followed by exposure to certain particles [36] or antineutrophil cytoplasmic antibody (ANCA) [37]. Recent studies proposed a breakthrough hypothesis about the significance of GSDMD in NETs release. ...
Renal fibrosis is a common consequence of various progressive nephropathies, including obstructive nephropathy, and ultimately leads to kidney failure. Infiltration of inflammatory cells is a prominent feature of renal injury after draining blockages from the kidney, and correlates closely with the development of renal fibrosis. However, the underlying molecular mechanism behind the promotion of renal fibrosis by inflammatory cells remains unclear. Herein, we showed that unilateral ureteral obstruction (UUO) induced Gasdermin D (GSDMD) activation in neutrophils, abundant neutrophil extracellular traps (NETs) formation and macrophage-to-myofibroblast transition (MMT) characterized by α-smooth muscle actin (α-SMA) expression in macrophages. Gsdmd deletion significantly reduced infiltration of inflammatory cells in the kidneys and inhibited NETs formation, MMT and thereby renal fibrosis. Chimera studies confirmed that Gsdmd deletion in bone marrow-derived cells, instead of renal parenchymal cells, provided protection against renal fibrosis. Further, specific deletion of Gsdmd in neutrophils instead of macrophages protected the kidney from undergoing fibrosis after UUO. Single-cell RNA sequencing identified robust crosstalk between neutrophils and macrophages. In vitro, GSDMD-dependent NETs triggered p65 translocation to the nucleus, which boosted the production of inflammatory cytokines and α-SMA expression in macrophages by activating TGF-β1/Smad pathway. In addition, we demonstrated that caspase-11, that could cleave GSDMD, was required for NETs formation and renal fibrosis after UUO. Collectively, our findings demonstrate that caspase-11/GSDMD-dependent NETs promote renal fibrosis by facilitating inflammation and MMT, therefore highlighting the role and mechanisms of NETs in renal fibrosis.
